1. Field of the Invention
The present invention relates to avian eggs, egg products, and egg fractions, methods for making the eggs, vaccines, and methods of using the eggs and egg products and fractions. The eggs and egg products contain factors effective for controlling cholesterol in serum and lipids in warm-blooded animals.
The factors are found in eggs and egg products and fractions obtained from animals that have been hyperimmunized. The factors have beneficial effects on the vascular system by lowering or maintaining the levels of serum cholesterol and lipid deposits in animals consuming the eggs or egg products or not elevating the levels of serum cholesterol and lipid deposits to those levels found in animals ingesting untreated eggs. The invention relates to the use of eggs, egg products, and egg fractions for the treatment of disorders of the vascular systems, such as vascular aging and arteriosclerosis. The beneficial effects are produced from ingestion of eggs, egg fractions, or egg products from animals hyperimmunized with specific bacterial antigens.
2. Description of the Background Art
Vascular Disorders
The normal vascular system of mammals, especially humans, includes all of the organs, such as the heart and the arteries, involved in blood transport and circulation. Two major disorders affect the vascular system in animals: arteriosclerosis and aging. Arteriosclerosis, a generic term for the thickening and hardening of the arterial wall, is responsible for the majority of deaths in the United States and most westernized societies. There are various types of arteriosclerosis, such as atherosclerosis, focal calcification, and arteriosclerosis. The changes associated with arteriosclerosis (of the various types) and aging are partly overlapping. (See, for example, Harrison's "Principles of Internal Medicine," 10th edition, pp. 1465-1475.)
The normal artery wall consists of three reasonably well-defined layers: the intima, the media, and the adventitia. The intima is a layer of endothelial cells lining the lumen of all arteries. The endothelial cells are attached to each other by a series of junctional complexes and also are attached to an underlying meshwork of loose connecting tissue, the basal lamina. The lining of endothelial cells forms a barrier that controls the entry of substances from the blood into the arterial wall. The media consists of smooth muscle cells arranged in either single layers or multiple layers. The outermost layer of the artery is the adventitia which is delimited by the external elastic lamina. This external coat consists of a loose interwoven mixture of thick bundles of collagen, elastic fibers of varying size, and a mixture of smooth muscle cells and fibroblasts.
Maintenance of the endothelial cell lining is critical. Endothelial cell turnover occurs at a slow rate but may accelerate in focal areas by changing patterns of flow along the vessel wall. Intact endothelial cells function to prevent clotting, partly by elaboration of prostacyclin that inhibits platelet function, thereby enhancing unimpeded flow of blood. When the lining is damaged, however, platelets adhere to it, in part as the result of production of a different class of prostaglandins, the thromboxanes, and form a clot. The ability of the arterial wall to maintain the integrity of its endothelium, prevent platelet aggregation, and ensure the nutrition of its middle portion may be the critical determinants of the arteriosclerotic process.
The major change that occurs with normal aging in the arterial wall is a slow symmetrical increase in the thickness of the intima. This results from an accumulation of small muscle cells. In the non-diseased artery wall, the lipid content, mainly cholesterol ester and phospholipid, also progressively increases with age. While most of the phospholipid in the normal artery wall appears to be derived from in situ synthesis, the cholesterol ester that accumulates with aging appears to be derived from plasma, as it contains principally linoleic acid, the major plasma cholesterol ester fatty acid. As the normal artery ages, smooth muscle cells and connective tissue accumulate in the intima, leading to progressive thickening of the layer, coupled with progressive accumulation of fatty acid, resulting in a gradual increase in the rigidity of the vessels. The larger arteries may become dilated, elongated, and porous, and aneurysms may form in areas of encroaching degenerating arteriosclerotic plaque.
By far, the leading cause of death in the United States above age 65, is atherosclerosis, the atheromatous form of arteriosclerosis. Atherosclerosis can be defined in broad terms as a vascular disease accompanied by dysregulation of cholesterol metabolism. This disease and its complications are the principal cause of mortality in the United States.
Progress has been made recently in understanding the basic biology of atherosclerosis (see, e.g., Hajjar, D. P. et al., FASEB Journal 6:2933-2941 (1992); Guyton, J. R. et al., American Journal of Pathology 141:925-936 (1992)). Yet, these basic data require clinical application if progress to be made toward treating and preventing this disease. The lesions are commonly classified as fatty streaks, fibrous plaques, and complicated lesions. The fatty streaks are characterized by an accumulation of lipid-filled smooth muscle cells and fibrous tissue in focal areas of the intima, and are stained distinctively by fat-soluble dyes. The lipid is mainly cholesterol oleate. Fibrous plaques are elevated areas of intima thickening, and will present the most characteristic lesion of advancing arteriosclerosis. They appear in the abdominal aorta, coronary arteries, and carotid arteries in the third decade, and increase progressively with age. Complicated lesions are calcified fibrous plaques containing various degrees of necrosis, thrombosis, and ulceration.
A number of "risk factors" have been identified in individuals who develop atherosclerosis. The risk factor concept implies that a person with at least one risk factor is more likely to develop a clinical atherosclerotic event and to do so earlier than a person with no risk factors. The presence of multiple risk factors further accelerates atherosclerosis. Among the reversible or partially reversible risk factors are hyperlipidemia (hypercholesterolemia and/or hypertriglyceridemia), hyperglycemia and diabetes mellitus, low levels of high-density lipoproteins in the presence of high concentrations of low-density lipoproteins, hypertension, obesity, and cigarette smoking.
As stated in Harrison's, supra (p. 1470), although the emergence of clinical consequences of atherosclerosis can be lessened, no convincing instance of regression or interruption of regression of atherosclerosis by removal or reversal of any single or group of risk factors has yet been proved in humans. The trend toward reduced smoking, lower cholesterol and fat consumption, reduction of body weight, and exercise programs have been helpful. Prevention, rather than treatment, however, is the goal of public health professionals. An effective program of prophylaxis has not yet been established, although enough is known to guide in both identification of high risk and development of measures to reduce the risk.
Among the risk factors referred to above that might be particularly well-suited to therapeutic treatment is hyperlipidemia. Although control of factors such as obesity and cigarette consumption depend, to a great degree, on the will and inclination of individual, if a reasonable method for lowering total serum cholesterol, low-density lipoprotein cholesterol and triglycerides in the circulation were provided, it would be suitable for treatment of a broad spectrum of individuals.
Because of the widespread distribution of vascular disorders such as arteriosclerotic disorders and the naturally occurring aging of the vascular system and its accompanying problems, a need exists for an effective method both for preventing and possibly treating these disorders. If a natural food product, such as milk or eggs, for example, could be obtained having anti-arteriosclerotic and anti-aging effects, it would be an easily administratible, readily available, safe therapeutic composition.
Products from Hyperimmunized Animals
It has been known in the prior art to produce milks having a variety of therapeutic effects. Beck, for example, has disclosed a milk containing antibody to Streptococcus mutans which has dental caries-inhibiting effects (Beck, U.S. Pat. No. 4,324,782). The milk is obtained by immunizing a cow with S. mutans antigen in two stages, and obtaining the therapeutic milk. Beck has also described milk having anti-inflammatory properties (U.S. Pat. No. 4,284,623) and anti-hypertensive properties (U.S. Pat. No. 4,879,110). Heinbach, U.S. Pat. No. 3,128,230, has described milk containing globulins of alpha, beta, and gamma components by inoculating a cow with antigenic mixtures. Petersen (U.S. Pat. No. 3,376,198 Canadian Patent 587,849) and Tunnah et al. (British Patent, 1,211,876) have also described antibody-containing milks.
U.S. Pat. Nos. 4,897,265 and 4,636,384 (Reissue No. 33,403) disclose a method for lowering blood lipid concentrations and thereby treating the aforementioned vascular disorders comprising feeding test animals and humans antibody-containing milk derived from cows maintained in a hyperimmune state by injections of polyvalent antigens derived from mixtures of bacteria.
It is known that various genera of the class Aves, such as chickens (Gallus domesticus), turkeys, and ducks, produce antibodies in their blood and in their eggs against factors which cause avian diseases, as well as against other antigens. For example, LeBacq-Verheyden et al., Immunology 27:683 (1974), and Leslie, G. A., et al., J. Med. 130:1337 (1969), have quantitatively analyzed immunoglobulins of the chicken. Polson, A., et al., Immunological Communications 9:495-514 (1980) immunized hens against several proteins and natural mixtures of proteins, and detected IgY antibodies in the yolks of the eggs. Fertel, R., et al., Biochemical and Biophysical Research Communications 102:1028-1033 (1981) immunized hens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al., Journal of Immunological Methods 46:63-68 (1981), provide a method of isolating egg yolk IgG for use in immunodiagnostics. Polson et al., Immunological Communications 9:475-493 (1980), describe antibodies isolated from the yolk of hens that were immunized with a variety of plant viruses.
Polson, U.S. Pat. No. 4,357,272, discloses the isolation of antibodies from the yolks of eggs derived from hyperimmunized hens. The hyperimmunization was elicited by repetitive injections into the hens of antigens represented by plant viruses, human IgG, tetanus antitoxin, snake antivenins, and Serameba antigens. Polson, U.S. Pat. No. 4,550,019, discloses the isolation from egg yolks of antibodies raised in the hen by hyperimmunization with immunogens having a molecular or particle weight of at least 30,000. The antigens used to hyperimmunize the chickens were selected from among plant viruses, human immunoglobulins, tetanus toxin, and snake venoms.
The present invention is a further development over the invention disclosed and claimed in U.S. application Ser. No. 577,804, filed Feb. 4, 1984, by Beck and Stolle, for "Heterologous Protein Antibody Formulation for Passive Immunization," now abandoned, and in U.S. Pat. No. 4,748,018, filed Jun. 19, 1984, by Stolle and Beck, for "Method of Passive Immunization of Mammals Using Avian Antibody." The entire disclosure of said patent is herein incorporated by reference.
In Ser. No. 577,804, abandoned there is disclosed a method of passive immunization of a mammal which comprises parenterally injecting a purified heterologous antibody obtained from the eggs of a domesticated fowl, which species has been immunized against an antigenic substance, and wherein the mammal has a history of consumption of eggs from such domesticated fowl. The invention disclosed in U.S. Pat. No. 4,748,018 expands on the concepts disclosed in U.S. Ser. No. 577,804, abandoned, in that administration of the egg antibody can be by any appropriate route, not only parenteral.
All of these references, however, relate only to immunoglobulins raised against various antigens by hyperimmunization and to the subsequent use of said immunoglobulins for either diagnostic procedures or homologous or heterologous passive immunization. No suggestion or speculation is made in these references either that the milk or eggs from hyperimmunized animals would have a beneficial effect on the vascular system by controlling serum cholesterol concentrations or lipid deposits in animals or that said hyperimmunized eggs could be consumed by humans and other animals without elevating serum lipid concentrations or not elevating them to the levels found in animals consuming normal eggs.